Centre for Advanced Macromolecular Design (CAMD), School of Chemistry, UNSW Sydney, Kensington, New South Wales 2052, Australia.
Department of Organic Chemistry, Bioorganic Chemistry, and Biotechnology, Faculty of Chemistry, Silesian University of Technology, B. Krzywoustego 4, Gliwice 44 100, Poland.
Biomacromolecules. 2021 Nov 8;22(11):4569-4581. doi: 10.1021/acs.biomac.1c00871. Epub 2021 Oct 7.
Trehalose is widely assumed to be the most effective sugar for protein stabilization, but exactly how unique the structure is and the mechanism by which it works are still debated. Herein, we use a polyion complex micelle approach to control the position of trehalose relative to the surface of glucose oxidase within cross-linked and non-cross-linked single-enzyme nanoparticles (SENs). The distribution and density of trehalose molecules in the shell can be tuned by changing the structure of the underlying polymer, poly(-[3-(dimethylamino)propyl] acrylamide (PDMAPA). SENs in which the trehalose is replaced with sucrose and acrylamide are prepared as well for comparison. Isothermal titration calorimetry, dynamic light scattering, and asymmetric flow field-flow fraction in combination with multiangle light scattering reveal that two to six polymers bind to the enzyme. Binding either trehalose or sucrose close to the enzyme surface has very little effect on the thermal stability of the enzyme. By contrast, encapsulation of the enzyme within a cross-linked polymer shell significantly enhances its thermal stability and increases the unfolding temperature from 70.3 °C to 84.8 °C. Further improvements (up to 92.8 °C) can be seen when trehalose is built into this shell. Our data indicate that the structural confinement of the enzyme is a far more important driver in its thermal stability than the location of any sugar.
海藻糖被广泛认为是最有效的蛋白质稳定剂糖，但它的结构究竟有多独特，以及它的作用机制仍存在争议。在此，我们使用聚离子复合物胶束的方法来控制海藻糖相对于交联和非交联单酶纳米颗粒（SENs）中葡萄糖氧化酶表面的位置。通过改变底层聚合物聚（[3-（二甲氨基）丙基]丙烯酰胺（PDMAPA）的结构，可以调节壳中海藻糖分子的分布和密度。还制备了用蔗糖和丙烯酰胺代替海藻糖的 SENs 进行比较。等温滴定量热法、动态光散射和非对称流场-流分离结合多角度光散射表明，有两到六个聚合物与酶结合。将海藻糖或蔗糖结合到酶表面附近对酶的热稳定性几乎没有影响。相比之下，将酶包封在交联聚合物壳内可显著提高其热稳定性，并将其解折叠温度从 70.3°C 提高到 84.8°C。当将海藻糖构建到该壳中时，可以看到进一步的改善（高达 92.8°C）。我们的数据表明，与任何糖的位置相比，酶的结构限制是其热稳定性的一个更重要的驱动因素。
